Annals of Botany 108: 381–390, 2011 doi:10.1093/aob/mcr142, available online at www.aob.oxfordjournals.org

Apomixis is not prevalent in subnival to nival of the European

Elvira Ho¨randl1,*, Christoph Dobesˇ2, Jan Suda3,4, Petr Vı´t3,4, Toma´sˇ Urfus3,4, Eva M. Temsch1, Anne-Caroline Cosendai1, Johanna Wagner5 and Ursula Ladinig5 1Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, A-1030 Vienna, , 2Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, A-1090 Vienna, Austria, 3Department of Botany, Faculty of Science, Charles University in Prague, CZ-128 01 Prague, Czech Republic, 4Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pru˚honice, Czech Republic and 5Institute of Botany, University of Innsbruck, A-6020 Innsbruck, Austria * For correspondence. E-mail [email protected]

Received: 2 December 2010 Returned for revision: 14 January 2011 Accepted: 28 April 2011 Published electronically: 1 July 2011

† Background and Aims High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown. † Methods The mode of reproduction in 14 species belonging to seven families was investigated via flow cyto- metric seed screen. The sampling comprised 12 species typical for nival to subnival communities of the European Alps without any previous information on apomixis ( atrata, alpina, Arabis caerulea, Erigeron uniflorus, Gnaphalium hoppeanum, Leucanthemopsis alpina, , Potentilla frigida, alpestris, R. glacialis, R. pygmaeus and bryoides), and two high-alpine species with apomixis reported from other geographical areas (Leontopodium alpinum and Potentilla crantzii). † Key Results Flow cytometric data were clearly interpretable for all 46 population samples, confirming the utility of the method for broad screenings on non-model organisms. Formation of endosperm in all species of was documented. Ratios of endosperm : embryo showed pseudogamous apomixis for Potentilla crantzii (ratio approx. 3), but sexual reproduction for all other species (ratios approx. 1.5). † Conclusions The occurrence of apomixis is not correlated to high altitudes, and cannot be readily explained by selective forces due to environmental conditions. The investigated species have probably other adaptations to high altitudes to maintain reproductive assurance via sexuality. We hypothesize that shifts to apomixis are rather connected to frequencies of polyploidization than to ecological conditions.

Key words: Apomixis, European Alps, endosperm, flow cytometric seed screen, high-altitude plants, polyploidy, sexual reproduction.

INTRODUCTION systems, flowering plants colonizing higher altitudes have to cope with conditions that are rather unfavourable for sexual Alpine biota often have a tendency to apomixis, the mode of reproduction. Colder climates, a short growing season and reproduction via asexually formed seed (Asker and Jerling, occasional frost during the reproductive period threaten a per- 1992). Apomictic plants show a higher abundance of seed than manent risk of seed loss, especially for plants that need a long their sexual relatives in higher altitudes and latitudes, in pre- time for seed development (e.g. Ladinig and Wagner, 2007; viously glaciated areas and in disturbed habitats Wagner et al., 2011). Apomixis, in contrast, is often associated (Bierzychudek, 1985; Ho¨randl, 2006; Ho¨randl et al., 2008). with an acceleration of developmental pathways via maturation This distribution pattern is most pronounced in cases of gameto- of embryo sacs before anthesis and pollination (e.g. Carman, phytic apomixis which involves the formation of an unreduced 1997; Grimanelli et al., 2001; Carman et al., 2011). Such pre- embryo sac and the development of an unreduced egg cell cocious development, known from alpine species of without fertilization. In contrast, the formation of embryos out Alchemilla (Fro¨hner, 1990) and from Hieracium alpinum of nucellar tissues (adventitious embryony) is more abundant (Skawin´ska, 1963), would allow for rapid seed formation in in tropical plants (Richards, 1997). a short growing season. Unfavourable weather conditions in The relationship of asexual reproduction to certain ecologi- high altitudes can reduce pollinator activities (e.g. Warren cal conditions has often been referred to selective forces by the et al., 1988; McCall and Primack, 1992), which may limit environment (Bell, 1982; Asker and Jerling, 1992). In the seed set for outcrossing plants (e.g. Mun˜oz and Arroyo, European Alps and in other comparable temperate mountain 2006). Therefore, reproductive assurance that is independent # The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 382 Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants of pollinators, as it is provided by apomixis or autogamy, between mountain systems (reviewed by Ho¨randl et al., would be advantageous in alpine habitats. Above the snowline, 2008; Ho¨randl, 2011), which infers that apomixis cannot be abiotic conditions become increasingly extreme in all these predicted for a certain region from studies on accessions in aspects. other mountain systems. Historical factors may have also promoted the occurrence of The reason for this lack of information on high-alpine plants apomixis in the Alps. Glaciations of the Pleistocene have is mostly due to methodological problems: experimental caused range fluctuations of species and may have brought pre- approaches (e.g. bagging and emasculation procedures) are dif- viously isolated species together, thereby increasing frequen- ficult to monitor in the field under high-alpine conditions, and cies of secondary contact hybridization and polyploidization do not discrimate between selfing and pollen-dependent apo- (Hewitt, 1996, 2004). Hybridization and/or polyploidy have mixis (pseudogamy; see Ho¨randl et al., 2008). This is critical been thought to cause the epigenetic and genetic changes because approx. 90 % of apomicts is in fact pseudogamous which would cause shifts to gametophytic apomixis (e.g. (Mogie, 1992). Reproductive tissues for studying embryo-sac Carman, 1997; Grimanelli et al., 2001; Koltunow and development before anthesis (e.g. after Herr, 1971, 1992)are Grossniklaus, 2003; Ho¨randl, 2009a, b). Moreover, uniparental often not accessible in high-alpine plants. In the lowlands, high- reproduction provides the additional advantage of enhanced alpine plants often do not flower in cultivation. Large-scale colonization abilities: a single individual can found a new progeny tests using molecular markers are often hampered by population (Baker’s law: Baker, 1967; Ho¨randl, 2006; low germination rates. For all these reasons, modes of reproduc- Ho¨randl et al., 2008). In the European Alps, the postglacial tion in high-alpine plants have remained largely unknown. retreat of glaciers offered opportunities for colonization by The recently developed method of flow cytometric seed screen apomictic plants (Cosendai and Ho¨randl, 2010). (FCSS; Matzk et al.,2000) can overcome these problems. FCSS The European Alps comprise ecologically distinct altitudi- measures DNA content in mature seeds and can therefore infer nal belts (Ozenda, 1988; Ellenberg and Leuschner, 2010): the DNA ploidy levels of embryo and endosperm, which the forest zone (montane zone) reaches 1300 m/1500 m a.s.l. allows the mode of apomixis to be determined (Krahulcova´ in the north/south, respectively, and is followed by the subal- and Rotreklova´, 2010). Sexual reproduction is characterized by pine zone (upper limits 2000 m/2200 m) with a mosaic of double fertilization, the egg cell and the central cell of the patchy forest, communities and pastures. The alpine embryo sac, and therefore the resulting ratio of embryo (n + n) zone (upper limits 2400 m/2600 m) is characterized by a to endosperm (2n + n) equals to 2 : 3. Gametophytic apomixis cover of vegetation by small and grassland. This veg- involves the formation of an unreduced embryo sac, and etation in the subnival zone (upper limits 2700/2900 m) the development of the unreduced egg cell without fertili- breaks up into isolated patches and is increasingly replaced zation. Seeds formed via apomixis, in contrast, have therefore by screes, permanent snow fields and rocks. The nival zone 1 : 2, 1 : 2.5 or 1 : 3 ratios of embryo (2n) to endosperm (2n + (from 2700/2900 m up to 4500 m) is characterized by glaciers 2n + 0, 2n + 2n + n or 2n + 2 n + n + n), depending on the and a predominance of cryptogams in patchy remnants of veg- contribution of sperm nuclei for endosperm fertilization. FCSS etation, with a drastic reduction of species richness of both was so far mostly used for traditional apomictic model organisms flowering plants and pollinators (Grabherr et al., 1995). (reviewed by Matzk, 2007), but has the potential to detect apo- Both ecological and historical factors would suggest that mixis in plants without embryological information (e.g. asexuality becomes more frequent in high altitudes because Heenan et al., 2002, 2003). of selective benefits. In fact, gametophytic apomixis has The main goal of this study was to screen for the occurrence been documented in many common species of the subalpine of apomixis in the high-alpine flora of the Alps and to test the and alpine zone of the European Alps (e.g. Alchemilla spp., utility of the FCSS methodology for non-model plants on Hieracium spp., Nardus stricta, Pilosella spp., Poa alpina, seeds collected in the wild. Fourteen candidate species were Taraxacum spp.; reviewed by Gustafsson, 1952, 1953; selected from seven plant families that are characteristic of Asker and Jerling, 1992; Ho¨randl, 2011). Some of them the subnival to the nival zone of the European Alps even dominate widespread alpine grassland communities (Schroeter, 1908; Ozenda, 1988; Grabherr et al., 1995), and (e.g. Nardus stricta, Poa alpina, Alchemilla spp.). flow cytometric seed screening on seeds collected in the Therefore, apomixis has been supposed to be a general repro- wild was conducted to test for the occurrence of apomixis. ductive strategy of alpine plants (e.g. Ko¨rner, 2003). It would be expected that apomixis should be even more abundant in species inhabiting subnival and nival zones than in species MATERIALS AND METHODS restricted to the subalpine and alpine zone because of the Materials increasingly extreme and stochastic climatic conditions. In arctic snowbeds, apomictic species become more frequent Seeds were collected in the wild during summer 2009 (for (Molau, 1993), which suggests a selective benefit to apomixis accessions see Table 1). Vouchers have been deposited in in habitats with a short vegetation period. However, the infor- the herbarium WU. In a few cases, plants in postfloral, but pre- mation on apomixis in high-alpine species of the European mature seed stage were transferred to the Botanical Garden in Alps was so far very scarce. From 12 species occurring Vienna, and seeds were collected there at maturity. Since over 4000 m a.s.l. as listed for example by Grabherr et al. embryo-sac formation and fertilization have happened before (1995), the mode of reproduction has never been studied. at the natural sites, this sampling strategy preserved the Moreover, apomictic taxa often show a geographical differen- mode of reproduction in the wild. Material was selected tiation of sexual and apomictic populations within and from the most frequent species of the subnival to nival zone TABLE 1. Materials used for the study

Altitude Taxon Collection no. Country* Province Locality, habitat type† (m a.s.l.) Co-ordinates Date Collector‡

Asteraceae Achillea atrata 9841 A Vorarlberg St Anton am Arlberg, Ulmer Hu¨tte; A, c 2273 47808′41.4′′N; 10812′31.6′′E01.08.2009 EH Achillea atrata 9862 A Tyrol Großglockner, Lucknerhu¨tte – Stu¨dlhu¨tte; A, sc 2357 47802′42.0′′N; 12841′21.0′′E11.08.2009 EH Achillea atrata 9866 A Tyrol Kals, Hohes Tor – Spo¨ttling; A, sc 2250 47801′31.0′′N; 12836′23.5′′E13.08.2009 EH Achillea atrata s.n. I Friuli-Venezia Julische Alps, Mt Kanin; A, c approx. 2000 46822′07′′N; 13828′41′′E07.09.2009 RN Erigeron uniflorus 9742 CH Valais Furkapass, Furkastock; R, sc 2638 46834′31.1′′N; 8824′48.5 ′′E26.07.2009 EH Erigeron uniflorus 9749 CH Valais Grimselpass, Sidelhorn; R, s 2528 46833′35.2′′N; 8819′07.3′′E27.07.2009 EH ′ . ′′ ′ . ′′ . . Ho

Erigeron uniflorus 9845 A Tyrol Verwall, Kuchenjo¨chli – Scheibler; R, s 2904 47803 23 3 N; 10813 20 9 E0508 2009 EH ¨ Erigeron uniflorus 9849 A Tyrol Verwall, Kuchenjo¨chli – Scheibler; R, s 2752 47803′16.0′′N; 10813′22.0′′E05.08.2009 EH randl Erigeron uniflorus 9854 A Tyrol Kals, Gorner; R, sc 2704 46859′09.7′′N; 12836′08.4′′E08.08.2009 EH Erigeron uniflorus 9859 A Tyrol Großglockner, Stu¨dlhu¨tte; R, sc 2847 47803′11.1′′N; 12840′53.5′′E11.08.2009 EH Gnaphalium hoppeanum 9838 A Tyrol St Anton am Arlberg, Kapall; B, c 2318 47809′06.8′′N; 10814′48.6′′E07.07.2009 EH — al. et Gnaphalium hoppeanum 9840 A Tyrol St Anton am Arlberg, Kapall; B, c 2270 47808′49.6′′N; 10814′59.3′′E07.07.2009 EH Leontopodium alpinum 9860 A Tyrol Grobglockner, Stu¨dlhu¨tte; R, sc 2862 47803′13.2′′N; 12840′54.5′′E11.08.2009 EH Leontopodium alpinum 9863 A Tyrol Kals, Muntanitzschneid; R, c 2288 47803′45.9′′N; 12836′43.9′′E08.08.2009 EH Leontopodium alpinum 9865 A Tyrol Kals, Du¨rrenfeldscharte; R, sc 2814 47802′32.4′′N; 12835′15.1′′E08.08.2009 EH plants nival to subnival in prevalent not is Apomixis Leontopodium alpinum 9877 A Lower Austria Schneeberg, near Dambo¨ckhaus; G, c 1865 47845′46.5′′N; 15849′49.7′′E30.08.2009 EH Leontopodium alpinum 9878 A Steiermark Raxalpe, near Raxkircherl; G, c 1820 47841′09.1′′N; 15842′13.2′′E09.09.2009 EH Leucanthemopsis alpina 9746 CH Valais Furkapass, Kl. Furkahorn; A, s 2739 46834′50.3′′N; 8824′45.0′′E26.07.2009 EH Leucanthemopsis alpina 9847 A Tyrol Verwall, Kuchenjo¨chli – Scheibler; A, s 2873 47803′21.7′′N; 10813′21.9′′E05.08.2009 EH Leucanthemopsis alpina 9861 A Tyrol Großglockner, Lucknerhu¨tte – Stu¨dlhu¨tte; A, sc 2475 47802′52.2′′N; 12841′25.1′′E11.08.2009 EH Brassicaceae Arabis caerulea 9829 CH Valais Furkapass, Blauberg; B, sc 2709 46834′01.7′′N; 8825′13.1′′E29.07.2009 EH Arabis caerulea 9839 A Tyrol St Anton am Arlberg, Kapall: B, c 2315 47809′06.1′′N; 10814′47.8′′E31.07.2009 EH Arabis caerulea 9875 A Lower Austria Schneeberg, Hackermulde; B, c 2013 47846′08.9′′N; 15848′27.1′′E30.08.2009 EH Oxyria digyna 9850 A Tyrol Verwall, Darmsta¨dter Hu¨tte – Saumspitze; A, s 2576 47802′48.7′′N; 10815′23.3′′E06.08.2009 EH Oxyria digyna s.n. A Tyrol O¨ tztal Alps, Mittelbergferner, glacier foreland; A, s 2850 46855′36.5′′N; 10852′55.0′′E22.09.2009 JW 9751 CH Valais Grimselpass, Sidelhorn; A, s 2516 46833′35.5′′N; 8819′08.9′′E27.07.2009 EH Androsace alpina 9844 A Tyrol Verwall, Kuchenjo¨chli – Scheibler; A, s 2904 47803′23.3′′N; 10813′20.9′′E05.08.2009 EH Androsace alpina 9852 A Tyrol Kals, Gorner; A, s 2506 46859′24.4′′N; 12835′57.7′′E08.08.2009 EH Androsace alpina 9856 A Tyrol Kals, Gorner; A, s 2680 46859′13.8′′N; 12836′11.1′′E08.08.2009 EH Androsace alpina 9858 A Tyrol Großglockner, Stu¨dlhu¨tte; A, s 2847 47803′11.1′′N; 12840′53.5′′E11.08.2009 EH Ranunculus alpestris 9835 A Tyrol St Anton am Arlberg, Kapall; B, c 2327 47809′07.8′′N; 10814′49.5′′E31.07.2009 EH Ranunculus alpestris 9836 A Tyrol St Anton am Arlberg, Kapall; B, c 2401 47809′19.0′′N; 10815′19.3′′E31.07.2009 EH Ranunculus alpestris 9876 A Lower Austria Schneeberg, Hackermulde; A, c 2030 47846′09.2′′N; 15848′25.1′′E08.08.2009 EH Ranunculus alpestris s.n. A Tyrol Innsbruck, Hafelekar; A, c 2300 47818′46.5′′N; 11823′05.0′′E Aug 2009 UL 889 F Hautes-Alpes Col de Galibier; not known 2500 4583′50.4′′N; 6824′28.8′′E 2009 Unknown§ Ranunculus glacialis 9750 CH Valais Goms, Sidelhorn; A, s 2525 46833′35.2′′N; 8819′07.3′′E07.07.2009 EH Ranunculus glacialis 9826 CH Valais Furkapass, Blauberg; A, sc 2643 46834′05.3′′N; 8825′15.2′′E29.07.2009 EH Ranunculus glacialis 9831 CH Valais Furkapass, Blauberg; A, sc 2838 46833′47.0′′N; 8825′13.1′′E29.07.2009 EH Ranunculus glacialis 9846 A Tyrol Verwall, Kuchenjo¨chli -Scheibler; A, s 2904 47803′23.3′′N; 10813′20.9′′E05.08.2009 EH Ranunculus glacialis 9857 A Tyrol Großglockner, Stu¨dlhu¨tte; A, sc 2847 47803′11.1′′N; 12840′53.5′′E11.08.2009 EH Ranunculus glacialis s.n. A Tyrol Stubai Alps, Schaufelferner, glacier foreland; A, s 2850 46859′15.8′′N. 11806′58.0′′E28.07.2009 UL Ranunculus pygmaeus 9855 A Tyrol Kals, Gorner; B, sc 2695 46859′08.0′′N; 12836′09.5′′E08.08.2009 EH Continued 383 384 Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants § ‡ after Schroeter (1908), Ozenda (1988) and Grabherr et al. (1995). Because of fuzzy borderlines between the upper alpine, subnival and nival zones, ‘high alpine’ is used here as an umbrella term for species occurring in these zones of the European Alps. We focused on species with insect pollina- 2009 EH 2009 EH 2009 UL 2009 Unknown . . . . tion to get insights into assumed selective effects of pollinator 07 08 09 07 . . . . limitation (thus excluding Poaceae and Cyperaceae). Diagnostic (characteristic and/or dominant) species of high- E15 E22 alpine plant communities (screes, snowbeds, exposed ridges; E29 ′′ ′′ ′′ 5 0 . . 1

. after Grabherr and Mucina, 1993) were selected to understand E01 09 58 ′′ ′ ′ 13

′ selective effects of environmental conditions on modes of 50 18 06 ′ 8 8 25

8 reproduction. Furthermore, an attempt was made to cover the 42 8 main geographical zones of the European Alps that are charac- N; 12 N; 8 N. 11 ′′ ′′ ′′ N; 6

1 0 8 terized by different geology and siliceous/calcareous bedrock; ′′ . . . 05 00 47 15

′ ′ ′ ′ see Table 1). The sampling comprises two groups. rmediate types. 33 59 06 02 8 8 8 8 (1) Typical subnival to nival species without any previous information on apomixis: Androsace alpina, Leucanthemopsis alpina, Ranunculus glacialis, Oxyria digyna and Saxifraga bryoides characterize high-alpine to nival siliceous screes, R. pygmaeus siliceous snowbeds. Achillea atrata, Arabis caeru- Altitude (m a.s.l.) Co-ordinates Date Collector lea, R. alpestris and Gnaphalium hoppeanum are diagnostic species of alpine to nival calcareous snowbeds; Erigeron uni- florus, Potentilla crantzii and P. frigida are typical of plant com- munities on exposed ridges. The mode of sexual versus apomictic reproduction has not yet been studied and so far apo- mixis is not even known in two of the respective families,

† Primulaceae and (Carman, 1997). However, apo- mixis occurs in other species of some of the respective genera [Achillea and Erigeron (Noyes, 2007), Ranunculus (Nogler, ¨ Continued 1984; Cosendai and Horandl, 2010) and Potentilla rl; R, sc 2382 47

¨ (Gustafsson, 1952, 1953)] and may be expected in the sampled 1. species because of taxonomic predispositions (e.g. Van Dijk and Vijverberg, 2005). ABLE

T (2) Species with apomixis reported from other areas: Potentilla crantzii and Leontopodium alpinum do have apo- mictic biotypes, as has been shown previously by cytological and embryological studies on materials outside the Alps (Sokolowska-Kulczycka, 1959; Czapik, 1961, 1962; Smith, 1963). Records of apomixis for L. alpinum in the Southern Alps (Maugini, 1962) need confirmation. Nevertheless, apo- mixis might be facultative in these species, and there is no information on the geographical distribution of modes of reproduction. Both species are not confined to the highest regions of the Alps but occur also in the alpine zone and are typical of plant communities on exposed ridges. A total of 46 populations were investigated, with an average of 3.4 populations per taxon. Multiple populations were ana- lysed for all but three species (Ranunculus pygmaeus, Potentilla frigida and Saxifraga bryoides). Apomixis is 838 F Hautes-Alpes Col de Lautaret; unknown 2400 45 9832 CH Valais Furkapass, Blauberg; A, sc 2838 46 s.n. A Tyrol Stubai Alps, Schaufelferner, glacier foreland; A, s 2850 46 9873 A Salzburg Großglockner, Mitterto heritable, and usually results in apomictic clones with one pre- dominant genotype and, if any, very low frequencies of deviat- ing individuals within a population. Predominance of apomictic plants, even in the case of facultative recombination, was confirmed by numerous population genetic studies (e.g. Gornall, 1999; Houliston and Chapman, 2004; Nybom et al., randl, RN, Roland Nitsche; UL, Ursula Ladinig; JW, Johanna Wagner.

¨ 2006; Paun et al., 2006; Thompson and Ritland, 2007; Barcaccia et al., 2007) and by progeny tests (e.g. Bicknell et al., 2003). Comprehensive FCSS studies suggest that

A, screes, glacier moraines;EH, B, E. snow beds; Ho R,Materials rocks from and the exposed Alpine ridges; Botanical G, Garden grassland of (patches); Lautaret, s, . siliceous bedrock; c, calcareous bedrock; sc, inte either sexuality or apomixis become rapidly fixed within a Potentilla frigida * A, Austria; CH, ; F, France; I, . Saxifraga bryoides Potentilla crantzii † ‡ § Potentilla crantzii Saxifragaceae TaxonRosaceae Collection no. Country* Province Locality, habitat type population (Aliyu et al., 2010). Therefore, the occurrence of Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants 385

TABLE 2. Results of flow cytometric seed screens

Protocol Population No. of Mean endosperm; Reproductive Species/family used no./locality individuals No. of seeds embryo peak ratio pathway

Asteraceae Achillea atrata 3 9841 1 127 1.48 Sexual 3 9862 2 20 (5 + 15) 1.47 Sexual 3 9866 1 20 1.48 Sexual 3 Julische Alps 1 25 1.5 Sexual Erigeron uniflorus 3 9742 1 72 1.48 Sexual 3 9749 1 20 1.49 Sexual 3 9845 2 40 (15 + 25) 1.49 Sexual 3 9849 1 35 1.47 Sexual 3 9854 5 61 (6 + 15 + 10 + 20 + 10) 1.48 Sexual 3 9859 1 20 1.45 Sexual Gnaphalium hoppeanum 3 9838 2 31 (21 + 10) 1.46 Sexual 3 9840 1 10 1.53 Sexual Leontopodium alpinum 3 9860 4 21 (6 + 6 + 5 + 4) 1.47 Sexual 3 9863 1 10 1.48 Sexual 3 9865 2 11 (6 + 5) 1.46 Sexual 3 9877 5 17 (7 + 6 + 2 + 1 + 1) 1.46 Sexual 3 9878 3 19 (12 + 5 + 2) 1.47 Sexual Leucanthemopsis alpina 3 9746 2 20 (10 + 10) 1.48 Sexual 3 9847 4 66 (12 + 11 + 27 + 16) 1.47 Sexual 3 9861 1 68 1.44 Sexual Brassicaceae Arabis caerulea 1 9829 1 3 1.52 Sexual 1 9839 2 13 (5 + 8) 1.55 Sexual 1 9875 1 3 1.53 Sexual Polygonaceae Oxyria digyna 2 9850 1 3 approx. 1.5* Sexual 3 Pitztal glacier 4 23 (7 + 6 + 5 + 5) 1.48 Sexual Primulaceae Androsace alpina 1 9751 1 4 1.54 Sexual 1 9844 1 3 1.55 Sexual 1 9852 1 3 1.56 Sexual 1 9856 1 3 1.55 Sexual 1 9858 1 3 1.56 Sexual Ranunculaceae Ranunculus alpestris 2 9835 1 5 1.58 Sexual 3 9835 3 15 (each 5) 1.59 Sexual 2 9836 1 5 1.67 Sexual 3 9836 3 15 (each 5) 1.62 Sexual 3 9876 2 10 (5 + 5) 1.62 Sexual 2 Hafelekar 9 45 (each 5) 1.63 Sexual 3 Hafelekar 3 15 (each 5) 1.58 Sexual Ranunculus glacialis 2 889 2 8 (3 + 5) 1.67 Sexual 3 889 1 9 1.64 Sexual 3 9750 2 20 (15 + 5) 1.72 Sexual 2 9826 2 10 (5 + 5) 1.82 ??? 3 9826 2 17 (11 + 6) 1.85 ??? 2 9831 1 5 1.78 Sexual 3 9831 3 26 (19 + 6 + 1) 1.75 Sexual 3 9846 6 37 (12 + 10 + 5 + 5 + 3 + 2) 1.80 ??? 2 9857 1 5 1.67 Sexual 3 9857 3 11 (5 + 3 + 3) 1.77 Sexual 2 Stubai glacier 2 10 (5 + 5) 1.64 Sexual Ranunculus pygmaeus 2 9855 1 5 1.69 Sexual Rosaceae Potentilla crantzii 1 838 1 6 2.96 Pseudogamous apomictic 1 9873 1 9 3.10 Pseudogamous apomictic Potentilla frigida 1 9832 1 1 1.50 Sexual Saxifragaceae Saxifraga bryoides 2 Stubai glacier 1 80 1.45 Sexual

For details on flow cytometric protocols, see Materials and methods. * Calculated from G2 peaks of both embryo and endosperm in immature seeds. 386 Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants apomixis can be assessed with high probability even on a FloMax software. The following design of seed analysis was single, randomly sampled individual of a population (e.g. usually adopted: one analysis of a single seed, five analyses Talent and Dickinson, 2007). [In contrast, the detection of of seed pairs, followed by the analyses of five pooled seeds rare recombinants within apomictic lineages requires large until the final seed number (see Table 2). sample sizes (Bicknell et al., 2003; Barcaccia et al., 2007; The embryo : endosperm ratios needed for the inference of Aliyu et al., 2010).] One individual for small populations the reproductive mode were calculated from the arithmetic and up to six for larger ones were collected. The number of means of the individual G0/G1 embryo and endosperm fluor- seeds analysed per species ranged from one in Potentilla escence peaks. The embryo/endosperm ratios of samples of frigida to 248 in Erigeron uniflorus, totalling 1143 seeds identical reproductive mode were averaged within accessions (see Table 2). for statistical presentation.

Flow cytometric seed screen The relative fluorescence intensity of nuclei from the A 300 embryo and endosperm were determined by flow cytometry 240 from single and/or pooled seeds and fruitlets. Materials were I II chopped with a razor blade and then specifically analysed according to the following protocols (see Table 2). 180 (a) At the Department of Pharmacognosy, University of Vienna, samples were prepared following Matzk et al. 120 (2000) using a one-step protocol using a slightly modified Number of nuclei seed buffer [5 mM MgCl2.6H2O, 85 mM sodium chloride, 60 100 mM Tris, 0.09 % Triton X-100, 6.1mM sodium citrate dihydrate, 1 mgmL21 DAPI (4′-6-diamidino-2-phenylindole)] 0 0 100 200 300 400 500 obtained from the Apomixis Working Group at the IPK, Relative fluorescence Gatersleben. The fluorescence intensity of 300–12 000 par- B 200 ticles was recorded using the Partec ploidy analyser PA (Partec GmbH., Mu¨nster, ) equipped with a 160 mercury arc lamp. I (b) At the Department of Systematic and Evolutionary 120 Botany, University of Vienna, seeds were chopped in Otto I . . buffer (0 1 M citric acid, 0 5 % Tween 20). After filtration 80 through a 30-mm mesh and incubation with RNase A 21 . Number of nuclei (0 15 mg mL )at378C for 30 min, propidium iodide (final 40 II concentration 50 mgmL21; Greilhuber et al., 2007) containing . M Otto II buffer (0 4 Na2HPO4.12H2O) was added. Staining 0 was carried out at 7 8C from 1 h up to overnight. For measure- 0 200 400 600 800 1000 ment a Partec CyFlow ML flow cytometer equipped with a Relative fluorescence diode-pumped solid state green laser (532 nm, 100 mW, F IG. 1. FCSS histogram from seeds formed by sexual reproduction. I, Cobolt Samba; Cobolt AB, Stockholm, Sweden) was used. Embryo nuclei; II, endosperm nuclei, with a ratio of approx. 2 : 3. (A) (c) At the Department of Botany, Faculty of Science, Androsace alpina (no. 9844); (B) Leontopodium alpinum (no. 9878). Charles University in Prague, seeds were analysed by DAPI (most samples) or propidium iodide (a few samples) flow cyto- metry following a simplified two-step protocol as described by Dolezˇel et al. (2007). Whole seeds (filled achenes selected I under the stereomicroscope) were chopped with or without a 640 tissue of internal reference standard (usually Pisum . sativum)in05 mL of ice-cold Otto I buffer. The crude suspen- 480 sion was filtered through a 42-mm nylon mesh and incubated at room temperature for 15 min. Nuclei were stained with 1 mL of Otto II buffer, supplemented with a fluorochrome and Counts 320 2-mercaptoethanol (2 mLmL21). As DNA-selective stains, 21 either DAPI (final concentration of 4 mgmL ) or propidium 160 ioidide + RNase IIA (both at final concentrations of 50 mg II III mL21) were used. After 10-min incubation at room tempera- 0 ture, fluorescence intensity of isolated nuclei was recorded 0 100 200 300 on a Partec ML or CyFlow SL cytometer equipped with a Fluorescence intensity power UV LED chip (365 nm) or a green solid-state laser F IG. 2. FCSS histogram from seeds of Potentilla crantzii (no. 9873) with (Cobolt Samba 532 nm, 100 mW), respectively, as an exci- apomictic pseudogamous reproduction. I, G1 embryo nuclei; II, G2 of the tation source. Flow histograms were evaluated using the embryo; III, G1 endosperm nuclei. Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants 387

RESULTS An essential prerequisite for FCSS is the presence of intact endosperm nuclei in mature seeds; this technique is thus not The FCSS analysis revealed clearly interpretable histograms applicable if endosperm in seeds is never formed (e.g. many with distinct embryo and endosperm peaks in 46 population Orchidaceae, Podostemaceae or Trapa natans) or is completely samples (Figs 1 and 2, Table 2 and Supplementary Data Fig. resorbed at maturity (e.g. Ceratophyllaceae, most Fabaceae, S1, available online). In most cases, single seeds or pooling some Fagaceae, Lythraceae and/or Asteraceae, including of two to five seeds provided sufficient amounts of tissue for Helianthus annuus; Black et al., 2006). Seeds of the latter revealing distinct peaks, except for Saxifraga bryoides, family usually have little endosperm (e.g. in Lactuca spp. whose seeds are so small that extensive pooling of seeds this nutrition tissue is formed by only one to a few layers of (approx. 80) was necessary. Calculations of peak ratios cells; Black et al., 2006); nevertheless, this amount is often usually show little variation among the samples of the same sufficient for FCSS to be successfully applied (e.g. species. In all species except R. glacialis, intraspecific vari- Taraxacum, Ma´rtonfiova´, 2006; Hieracium, A. Krahulcova´ ation was below 6 % (Table 2). et al., Pru˚honice, unpubl. res.). Similarly, all Asteraceae All subnival to nival species with unknown mode of repro- species analysed in the present study had endospermous duction and no apomictic relatives (Androsace alpina, Arabis seeds as shown by a distinct peak corresponding to endosperm caerulea, Gnaphalium hoppeanum, Leucanthemopsis alpina, nuclei (see Fig. 1B and Supplementary Data Fig. S1). FCSS on Oxyria digyna and Saxifraga bryoides) showed exclusively other Asteraceae species is desirable to test the validity of the ratios of embryo to endosperm of 2 : 3 as is typical for conventional assumption that mature seeds of this family gen- double fertilization [a reduced egg cell fertilized by one erally lack endosperm. Another potential problem of FCSS sperm nucleus, n + n, and the two polar nuclei (or the may be the histogram interpretation, especially when pooled central cell nucleus) fertilized by the other sperm nucleus, seeds are analysed. Mixed sexual and apomictic seed 2n + n; Fig. 1]. This result confirms sexual reproduction in samples would reveal distinct endosperm peaks at the expected these species. In Oxyria digyna, embryo tissue underwent ratios to the embryo, but not an intermediate ratio (e.g. endoreduplication, resulting in a high peak of nuclei with 4C Barcaccia et al., 2007; Aliyu et al., 2010). Since just one endo- DNA content. sperm peak was observed in R. glacialis, a mixed sexual– Also the species with apomixis known in the same apomictic system can be excluded as interpretation. It may (Achillea atrata, Erigeron uniflorus, Potentilla frigida, be difficult to set a reasonable threshold for discrimination Ranunculus alpestris, R. glacialis and R. pygmaeus) were all of sexual versus apomictic seeds if pronounced, and more or exclusively sexual (Table 2). In R. glacialis, peak ratios were less continuous, variation in peak ratios is observed, such as rather variable (intraspecific variation nearly 13 %) and in Ranunculus glacialis (Table 2). In this species, endosperm : reached values from 1.64 up to 1.85, but always remained embryo peak ratios were always above 1.5 (i.e. the value clearly below 2.0 which would be indicative of an unreduced typical for sexual reproduction) but below 2.0 (i.e. the value embryo sac. Plants with peak ratios below 1.8 are considered typical for autonomous apomixis). Because the peak ratios in as sexual and those with peak ratios above this arbitrary other sexually reproducing buttercups are known to vary con- threshold (1.80–1.85) as having an uncertain mode of repro- sistently between 1.5 and 1.8(Cosendai and Ho¨randl, 2010; duction (populations 9826 and 9846; Table 2). E. Ho¨randl and D. Hojsgaard, unpubl. res.; see also In the two alpine species with previously reported apomixis, Table 2), ratios below 1.8 were regarded to be indicative of only Potentilla crantzii showed apomictic seed formation with sexual reproduction. The mode of reproduction in samples around three times the DNA content of the endosperm com- with peak ratios exceeding this arbitrary threshold should be pared with the embryo (Fig. 2 and Table 2). This result indi- investigated using other methodological approaches such as cates an unreduced embryo sac connected to pseudogamous dissecting techniques. Whether the observed variation in the apomixis, whereby either both sperm nuclei or one unreduced endosperm : embryo peak ratios has technical backgrounds or sperm nucleus must have fertilized the central cell (2n + 2n + reflects natural variation in the DNA content of gametes, n + n or 2n + 2n + 2n). In contrast, all five accessions of remains to be studied. However, it should be noted that Leontopodium alpinum from the Alps clearly showed sexual highly comparable peak ratios for a particular population of reproduction. R. glacialis were obtained in two different laboratories and using different protocols (Table 2). Natural variation in DNA content within cytotypes, as it was observed in populations of diploid R. kuepferi (Cosendai and Ho¨randl, 2010), might DISCUSSION influence gamete ratios. Another possibility is differential This study confirms the broad applicability of FCSS for the expression of secondary metabolites, or differential DNA determination of the mode of reproduction in high-alpine degeneration due to drying of tissues in embryo and endo- plant species. Further sampling and routine screening can sperm. Autonomous apomixis or haploid parthenogenesis establish geographical patterns of modes of reproduction, as were considered unlikely as no value reached the typical has been shown by Cosendai and Ho¨randl (2010) in the ratio of 2. alpine species R. kuepferi. Potential drawbacks are that Despite theoretical evolutionary advantages, gametophytic FCSS cannot detect adventitious embryony and is further not apomixis turns out to be very rare in plants at extremely applicable to rare forms of gametophytic development with high elevations. This confirms the opinion of Gustafsson four-nucleate embryo sacs, where only one polar nucleus is (1952, 1953) that frequencies of apomixis decline from high formed (e.g. Carman, 1997). to very high altitudes. The selective benefits of uniparental 388 Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants reproduction for colonization, pollinator-independence and Pilosella spp., Poa alpina, Taraxacum spp.; Ho¨randl, 2011). rapid development seem to be not strong enough to establish Plants from lower latitudes that are not adapted to cold temp- apomixis as a frequent trait. Facultative selfing might eratures may undergo more frequently polyploidization and provide an alternative that is functionally easier to establish shifts to apomixis during colonization of higher elevations. (Ho¨randl, 2006). From the species investigated here, selfing In contrast, the high-elevation plants studied here do not has been documented for Gnaphalium hoppeanum and show a pronounced tendency towards polyploidy, which Arabis caerulea, while Achillea atrata is predominantly out- again may be explained by specific adaptations to cold temp- crossing (Scheffknecht et al., 2007). The two selfing species eratures. The present study on high-alpine plants confirms occur in snow-beds, where extremely short vegetation that apomixis is not increased in frequencies by selective periods may favour uniparental reproduction. Selfing and apo- forces of environmental conditions if functional requirements mixis, however, are usually rather alternative strategies of uni- for shifts to apomixis are not met. This supports the hypothesis parental reproduction (Ho¨randl, 2010). Predominant by Ho¨randl (2009b) that asexual reproduction is limited by outcrossing appears to be a competitive strategy in subnival functional constraints for shifts from sexuality to apomixis. plants as well and has been confirmed, for example, on Ranunculus glacialis (Wagner et al., 2010) and on Saxifraga bryoides (Ladinig and Wagner, 2007). For the other species, SUPPLEMENTARY DATA detailed studies on breeding systems and reproductive Supplementary data are available online at www.aob.oxford- success are largely missing. We hypothesize that these high- journals.org and consist of Fig. S1: representative FCSS histo- alpine specialists are so well adapted to the harsh and stochas- grams of seeds of four Asteraceae species with sexual tic environmental conditions that a shift to apomixis would not reproduction. provide a strong selective advantage. No apomixis was found in those species where the trait has been observed in other species of the genus. However, the ACKNOWLEDGEMENTS present results clearly confirm that gametophytic apomixis is We thank Jana Rauchova´ for her help with pilot flow cytometric connected to polyploidy. All the observed sexual species are analyses and Roland Nitsche (Vienna) for collection of a sample diploid except for Androsace alpina (2n ¼ 4x ¼ 32; Dobesˇ from the south-eastern Alps. This work was supported by the and Vitek, 2000). Leucanthemopsis alpina has diploid and tet- Austrian Academy of Sciences, Commission for Interdiscipli- raploid cytotypes in the Alps (Watanabe, 2011). In contrast, nary Ecological Studies (KIO¨ S grant no. 2009/03) and the species with recorded or here observed apomixis FWF Austrian Science Fund (grant number I310), both to E.H. (Leontopodium alpinum and Potentilla crantzii, respectively) Further support was provided by the Academy of Sciences of are all polyploid. Potentilla crantzii comprises several poly- the Czech Republic (grant number AV0Z60050516) and the ploid cytotypes with 2n ¼ 28, 42 and 49 (Dobesˇ and Vitek, Ministry of Education, Youth and Sports of the Czech 2000). The present observation of apomixis in the plants Republic (grant number MSM0021620828) to J.S. from the Alps confirms previous studies on material from the British Isles, whereas the species is sexual in the Carpathians (Czapik, 1961, 1962; Smith, 1963). Leontopodium alpinum LITERATURE CITED in the Tatry mountains is polyploid with 2n ¼ 4x ¼ 52 Aliyu OM, Schranz ME, Sharbel TF. 2010. Quantitative variation for apo- (Murı´n and Paclova´, 1979). For edelweiss, apomixis has mictic reproduction in the genus Boechera (Brassicaceae). American been reported in material from the Carpathians Journal of Botany 97: 1719–1731. Asker SE, Jerling L. 1992. Apomixis in plants. Boca Raton, FL: CRC Press. (Sokolowska-Kulczycka, 1959), while studies on material Baker HG. 1967. Support for Bakers law – as a rule. Evolution 21: 853–856. from the southern Alps suggested facultative apomixis Barcaccia G, Arzenton F, Sharbel T, Varotto S, Parrini P, Lucchin M. (Maugini, 1962). The present data show sexual reproduction 2007. Genetic diversity and reproductive biology in ecotypes of the facul- in five accessions of the eastern Alps. These cases may tative apomict Hypericum perforatum L. Heredity 96: 322–334. reflect a geographical differentiation of apomictic and sexual Bell G. 1982. The masterpiece of nature: the evolution and genetics of sexu- ality. Berkeley, CA: California Press. cytotypes between the Alps and the Tatry as also observed Bicknell RA, Lambie SC, Butler RC. 2003. Quantification of progeny classes in Hieracium (Mra´z et al., 2009). in two facultatively apomictic accessions of Hieracium. Hereditas 138: Gametophytic apomixis has been observed as a rare trait in 11–20. diploid, otherwise sexual plants (e.g. Boechera, Kantama Bierzychudek P. 1985. Patterns in plant parthenogenesis. Experientia 41: 1255–1264. et al., 2007; Paspalum, Siena et al., 2008). However, establish- Black M, Bewley JD, Halmer P. 2006. The encyclopedia of seeds. ment of gametophytic apomixis is usually connected to poly- Wallingford, UK: CABI. ploidy (Grimanelli et al., 2001; Koltunow and Grossniklaus, Carman JG. 1997. Asynchronous expression of duplicate genes in angios- 2003). Polyploidization happens more frequently in colder cli- perms may cause apomixis, bispory, tetraspory, and polyembryony. mates, as cold temperatures can trigger the formation of unre- Biological Journal of the Linnean Society 61: 51–94. Carman JG, Jamison M, Elliott E, Dwivedi KK, Naumova TN. 2011. duced gametes (Ramsey and Schemske, 1998). In this respect, Apospory appears to accelerate onset of meiosis and sexual embryo sac frequencies of polyploidization in a certain taxon might be formation in sorghum ovules. BMC Plant Biology 11: 9. doi:10.1186/ more important for the establishment of apomixis than selec- 1471-2229-11-9. tive forces of ecological conditions. In fact, apomixis in the Cosendai A-C, Ho¨randl E. 2010. Cytotype stability, facultative apomixis and geographical parthenogenesis in Ranunculus kuepferi (Ranunculaceae). Alps occurs in species of the more moderate subalpine and Annals of Botany 105: 457–470. alpine grassland zone, sometimes extending to the forest Czapik R. 1961. Embryological studies in the genus Potentilla zone (Alchemilla spp., Hieracium spp., Nardus stricta, L. I. P. crantzii. Acta Biologica Cracoviensia, Ser. Botanica 4: 97–119. Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants 389

Czapik R. 1962. Badania embriologiczne nad rodzajem Potentilla L. III. Koltunow A, Grossniklaus U. 2003. Apomixis, a developmental perspective. Mieszance miedzy P. crantzii i P. arenaria [Embryological studies in Annual Review of Plant Biology 54: 547–574. the genus Potentilla L. III. Hybrids between P. crantzii and Ko¨rner Ch. 2003. Alpine plant life: functional plant ecology of high mountain P. arenaria]. Acta Biologica Cracoviensia, Ser. Botanica 5: 43–61. ecosystems. Berlin: Springer. Dobesˇ C, Vitek E. 2000. Documented chromosome number checklist of Krahulcova´ A, Rotreklova´ O. 2010. Use of flow cytometry in research on Austrian vascular plants. Vienna: Museum of Natural History. apomictic plants. Preslia 82: 23–39. Dolezˇel J, Greilhuber J, Suda J. 2007. Estimation of nuclear DNA content in Ladinig U, Wagner J. 2007. Timing of sexual reproduction and reproductive plants using flow cytometry. Nature Protocols 2: 2233–2244. success in the high-mountain plant Saxifraga bryoides L. Plant Biology 9: Ellenberg H, Leuschner C. 2010. Vegetation Mitteleuropas mit den Alpen, 683–693. 6th edn. Stuttgart: Ulmer. McCall C, Primack RB. 1992. Influence of flower characteristics, weather, Fro¨hner S. 1990. Alchemilla. In: Conert HJ, et al. eds. Illustrierte Flora von time of day, and season on insect visitation rates in three plant commu- Mitteleuropa, Vol. IV 2B. edn 3. Berlin: Parey, 13–242. nities. American Journal of Botany 79: 434–442. Gornall RJ. 1999. Population genetic structure in agamospermous plants. In: Ma´rtonfiova´ L. 2006. Possible pathways of the gene flow in Taraxacum sect. Hollingsworth PM, Bateman RM, Gornall RJ. eds. Molecular systematics Ruderalia. Folia Geobotanica 41: 183–201. and plant evolution. London: Taylor & Francis, 118–138. Matzk F. 2007. Reproduction mode screening. In: Dolezˇel J, Greilhuber J, ¨ Grabherr G, Mucina L. eds. 1993. Die Pflanzengesellschaften Osterreichs. Suda J. eds. Flow cytometry with plant cells: analysis of genes, chromo- Teil II. Natu¨rliche waldfreie Vegetation. Jena: G. Fischer. somes, and genomes. Weinheim: Wiley–VCH, 131–152. Grabherr G, Gottfried M, Gruber A, Pauli H. 1995. Patterns and current Matzk F, Meister A, Schubert I. 2000. An efficient screen for reproductive changes in alpine plant diversity. In: Chapin FS III, Ko¨rner C. eds. pathways using mature seeds of monocots and dicots. The Plant Arctic and alpine biodiversity. Ecological Studies 113. Berlin: Springer, Journal 21: 97–108. 167–181. Maugini E. 1962. Morfologia fiorale, embriologia ed embryogenesis in Greilhuber J, Temsch EM, Loureiro JCM. 2007. Nuclear DNA content Leontopodium alpinum Cass. var. typicum Fiori e Paoletti. Giornale measurement. In: Dolezˇel J, Greilhuber J, Suda J. eds. Flow cytometry Botanico Italiano 69: 1–18. with plant cells: analysis of genes, chromosomes, and genomes. Mogie M. 1992. The evolution of asexual reproduction in plants. London: Weinheim: Wiley-VCH, 67–101. Chapman and Hall. Grimanelli D, Leblanc O, Perotti E, Grossniklaus U. 2001. Molau U. 1993. Realtionships between phenology and life history strategies in Developmental genetics of gametophytic apomixis. Trends in Genetics tundra plants. Arctic and Alpine Research 25: 391–402. 17: 597–604. Mra´z P, Chrtek J, Sˇingliarova´ B. 2009. Geographical parthenogenesis, Gustafsson A. 1952. Apomixis in higher plants. Part I. The mechanisms of genome size variation and pollen production in the arctic-alpine species apomixis. Lunds Universitets Arsskrift 42: 1–66. Hieracium alpinum. Botanica Helvetica 119: 41–51. Gustafsson A. 1953. Apomixis in higher plants. Part II. The causal aspects of Mun˜oz A, Arroyo MTK. 2006. Pollen limitation and spatial variation of Lunds Universitets Arsskrift 43 apomixis. : 71–178. reproductive success in the insect-pollinated shrub Chuquiraga oppositi- Heenan PB, Dawson MI, Bicknell RA. 2002. Evidence for apomictic seed folia (Asteraceae) in the Chilean Andes. Arctic, Antarctic and Alpine formation in Coprosma waima (Rubiaceae). New Zealand Journal of Research 38: 608–613. Botany 40: 347–355. Murı´n A, Paclova´ L. 1979. IOPB chromosome number reports LXIV. Taxon Heenan PB, Molloy BPJ, Bicknell RA, Luo C. 2003. Levels of apomictic 28: 403–405. and amphimictic seed formation in a natural population of Coprosma Nogler GA. 1984. Genetics of apospory in apomictic Ranunculus auricomus. robusta (Rubiaceae) in Riccarton Bush, Christchurch, New Zealand. V. Conclusion. Botanica Helvetica 94: 411–423. New Zealand Journal of Botany 41: 287–291. Noyes RD. 2007. Apomixis in the Asteraceae: diamonds in the rough. Herr JM. 1971. A new clearing-squash technique for the study of Functional Plant Science and Biotechnology 1: 207–222. ovule development in angiosperms. American Journal of Botany 58: 785–790. Nybom H, Esselink GD, Werlemark G, Leus L, Vosman B. 2006. Unique Herr JM. 1992. Recent advances in clearing techniques for study of ovule genomic configuration revealed by microsatellite DNA in polyploid and female gametophyte development. Angiosperm pollen and ovules. dogroses, Rosa sect. Caninae. Journal of Evolutionary Biology 19:635–648. New York: Springer, 149–154. Ozenda P. 1988. Die Vegetation der Alpen im europa¨ischen Gebirgsraum. Hewitt GM. 1996. Some genetic consequences of ice ages, and their role in Stuttgart: Gustav Fischer Verlag. divergence and speciation. Biological Journal of the Linnean Society Paun O, Greilhuber J, Temsch E, Ho¨randl E. 2006. Patterns, sources and 58: 247–276. ecological implications of clonal diversity in apomictic Ranunculus car- Hewitt GM. 2004. Genetic consequences of climatic oscillations in the paticola (Ranunculus auricomus complex, Ranunculaceae). Molecular Quaternary. Philosophical Transactions of the Royal Society of London, Ecology 15: 897–910. Ser. B: Biological Sciences 359: 183–195. Ramsey J, Schemske DW. 1998. Pathways, mechanisms, and rates of poly- Ho¨randl E. 2006. The complex causality of geographical parthenogenesis. ploid formation in flowering plants. Annual Review of Ecology and New Phytologist 171: 525–538. Systematics 29: 467–501. Ho¨randl E. 2009a. Geographical parthenogenesis: opportunities for asexual- Richards AJ. 1997. Plant breeding. London: Chapman & Hall. ity. In: Scho¨n I, Martens K, Van Dijk P. eds. Lost sex. Heidelberg: Scheffknecht S, Dullinger S, Grabherr G, Hu¨lber K. 2007. Mating Springer Verlag, 161–186. systems of snowbed plant species of the northeastern calcareous Alps Ho¨randl E. 2009b. A combinational theory for maintenance of sex. Heredity of Austria. Acta Oecologica – International Journal of Ecology 31: 103: 445–457. 203–209. Ho¨randl E. 2010. The evolution of self-fertility in apomictic plants. Sexual Schroeter C. 1908. Das Pflanzenleben der Alpen.Zu¨rich: A. Raustein. Plant Reproduction 23: 73–86. Siena LA, Sartor ME, Espinoza F, Quarin CL, Ortiz JPA. 2008. Genetic Ho¨randl E. 2011. Evolution and biogeography of alpine apomictic plants. and embryological evidences of apomixis at the diploid level in Taxon 60: 390–402. Paspalum rufum support recurrent auto-polyploidization in the species. Ho¨randl E, Cosendai A-C, Temsch E. 2008. Understanding the geographic Sexual Plant Reproduction 21: 205–215. distributions of apomictic plants: a case for a pluralistic approach. Skawin´ska R. 1963. Apomixis in Hieracium alpinum L. Acta Biologica Plant Ecology and Diversity 2: 309–320. Cracoviensia, Ser. Botanica 5: 7–14. Houliston HJ, Chapman HM. 2004. Reproductive strategy and population Smith GL. 1963. Studies in Potentilla L. II. Cytological aspects of apomixis in genetic variability in the facultative apomict Hieracium pilosella P. crantzii (Cr.) Beck ex Fritsch. New Phytologist 62: 283–300. (Asteraceae). American Journal of Botany 91: 37–44. Sokolowska-Kulczycka A. 1959. Apomiksja u Leontopodium alpinum CASS Kantama L, Sharbel TF, Schranz ME, Mitchell-Olds T, de Vries S, de [Apomixis in Leontopodium alpinum CASS]. Acta Biologica Jong H. 2007. Diploid apomicts of the Boechera holboellii complex Cracoviensia, Ser. Botanica 2: 51–63. display large-scale chromosome substitutions and aberrant chromosomes. Talent N, Dickinson TA. 2007. Endosperm formation in aposporous Proceedings of the National Academy of Sciences of the United States of Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): parallels to America 104: 14026–14031. Ranunculaceae and Poaceae. New Phytologist 173: 231–249. 390 Ho¨randl et al. — Apomixis is not prevalent in subnival to nival plants

Thompson SL, Ritland K. 2007. A novel mating system analysis for modes of Wagner J, Ladinig U, Steinacher G, Larl I. 2011. From the flower self-oriented mating applied to diploid and polyploid arctic Easter daisies bud to the mature seed: timing and dynamics of flower and seed (Townsendia hookeri). Heredity 97: 119–126. development in high-mountain plants. In: Lu¨tz C. ed. Plants in Van Dijk PJ, Vijverberg K. 2005. The significance of apomixis in the evol- alpine regions: cell physiology of adaption and survival strategies. ution of the angiosperms: a reappraisal. In: Bakker F, Chatrou L, New York: Springer. Gravendeel B, Pelser PB. eds. Plant species-level systematics: new per- Warren SD, Harper KT, Booth GM. 1988. Elevational distribution of insect spectives on pattern and process. Ruggell, Liechtenstein: Gantner pollinators. American Midland Naturalist 120: 325–330. Verlag, 101–116. Watanabe K. 2011. Index to chromosome numbers in Asteraceae. Kobe Wagner J, Steinacher G, Ladinig U. 2010. Ranunculus glacialis L.: success- University Library Digital Archive. http://www.lib.kobe-u.ac.jp/ ful reproduction at the altitudinal limits of higher plant life. Protoplasma products/e-index.html (accessed January 2011). 243: 117–128.